Abstract
Purpose :
Corneal hypoxia can result from conditions such as diabetes and corneal infections. Our previous data indicate that hypoxia leads to delayed wound healing compared to controls. The aim of this study is to examine basal lamina compositional changes and to determine how these alterations affect focal adhesion (FA) turnover and the machinery involved in migration.
Methods :
An ex vivo corneal organ culture model was employed to examine cell migration and wound healing. Injuries were made using a 3 millimeter trephine and corneas were incubated in normoxia or hypoxia and fixed at different timepoints for immunohistochemistry (IHC). An in vitro scratch wound model was employed with human corneal limbal epithelial (HCLE) cells. After injury, cultures were fixed for IHC or probed for western blot analysis. Live cell assays were performed to examine HCLE migration on fibronectin (FN) or collagen IV (coll IV)-coated polyacrylamide gels covering a range of stiffnesses. HCLEs were transfected with mCherry-vinculin and seeded onto gels of varying stiffness to determine alterations in FA turnover along wound edges. Traction force microscopy (TFM) was employed to study changes in force dynamics of HCLES and corneal epithelial sheets on FN-coated substrates.
Results :
Wounded corneas exposed to hypoxia present with alterations in both the morphology of the leading edge and the composition of the basal lamina. Under hypoxia, the leading edge is blunt and localization of fibronectin appears to be intracellular. Cell morphology, size, and motility is affected by the change in substrate stiffness. Stiffness also played a role in mediating characteristics of FAs present in organ and cell culture. Expression and localization of vinculin Y1065, paxillin Y118, and vinculin Y822 are altered in our organ culture model and in vitro system. Western blot analysis shows that after wounding, phosphorylation is greatest at the initial stages of migration. Furthermore, as substrate stiffness increased, FA count along the leading edge decreases, FA size increases, and changes in FA turnover and force is observed.
Conclusions :
Using model systems and live cell assays we can investigate impaired wound healing in various pathologies involving corneal hypoxia. Determining alterations in basal lamina composition and stiffness and characterizing the changes in regulation of FA proteins, will allow for the development of treatment modalities.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.